Potential therapeutic biomolecules of hymenopteran venom against SARS-CoV-2 from Egyptian patients

The therapeutic potential of insect-derived bioactive molecules as anti-SARS-CoV-2 agents has shown promising results. Hymenopteran venoms, notably from Apis mellifera (honeybee) and Vespa orientalis (oriental wasp), were examined for the first time in an in vitro setting for their potential anti-COVID-19 activity. This assessment utilized an immunodiagnostic system to detect the SARS-CoV-2 nucleocapsid antigen titer reduction. Further analyses, including cytotoxicity assays, plaque reduction assays, and in silico docking-based screening, were performed to evaluate the efficacy of the most potent venom. Results indicated that bee and wasp venoms contain bioactive molecules with potential therapeutic effects against SARS-CoV-2.Nevertheless, the wasp venom exhibited superior efficacy compared to bee venom, achieving a 90% maximal (EC90) concentration effect of antigen depletion at 0.184 mg/mL, in contrast to 2.23 mg/mL for bee venom. The cytotoxicity of the wasp venom was assessed on Vero E6 cells 48 h post-treatment using the MTT assay. The CC 50 of the cell growth was 0.16617 mg/mL for Vero E6 cells. The plaque reduction assay of wasp venom revealed 50% inhibition (IC50) at a 0.208 mg/mL concentration. The viral count at 50% inhibition was 2.5 × 104 PFU/mL compared to the initial viral count of 5 × 104 PFU/mL. In silico data for the wasp venom revealed a strong attraction to binding sites on the ACE2 protein, indicating ideal interactions. This substantiates the potential of wasp venom as a promising viral inhibitor against SARS-CoV-2, suggesting its consideration as a prospective natural preventive and curative antiviral drug. In conclusion, hymenopteran venoms, particularly wasp venom, hold promise as a source of potential therapeutic biomolecules against SARS-CoV-2. More research and clinical trials are needed to evaluate these results and investigate their potential for translation into innovative antiviral therapies.

indicating a wide therapeutic window.However, subsequent analysis revealed that WV exerted no inhibitory effect on the replication of established HCV infections.Therefore, the mechanism of action appears to be limited to the early stages of viral entry.This finding suggests that WV may offer potential as a prophylactic agent against HCV, warranting further investigation 23 .Similarly, the mastoparan-derived peptide MP7-NH2 exhibited potent direct inactivation of various enveloped viruses.This selectivity for enveloped viruses suggests that MP7-NH2 targets a component of the viral envelope, likely the lipid bilayer.Furthermore, pre-treatment of cells with MP7-NH2 failed to reduce the subsequent recovery of infectious virus, further supporting the hypothesis that its primary mechanism of action is direct inactivation of extracellular virions 24 .
The in vitro antiviral properties of insect venom against SARS-CoV-2 have not been reported.Nevertheless, in silico studies focusing on bee venom were conducted, suggesting the need for further in vitro and in vivo investigations 25 .
This work aimed to search for antiviral substitutes targeting the S protein that are low or completely free of diverse effects, which is an urgent need.In this context, Hymenoptera venom, specifically bee and wasp venom, encapsulates diverse exotic constituents, implying an extensive reservoir of potential antiviral agents, as indicated by prior literature documenting their antiviral activity.Moreover, the control of SARS-CoV-2 reproduction and inhibition of its protein synthesis was investigated by assessing the antiviral properties of Hymenoptera venom, explicitly targeting the inhibitory activity on nucleocapsid and spike viral proteins in vitro.

Results
Evaluation of the antiviral activity of Hymenoptera venom against SARS-CoV-2 using Ortho VITROS® Immunodiagnostic System: Nucleocapsid protein antigens from SARS-CoV-2 were quantitatively detected in nasopharyngeal swab (NP) specimens using a fully automated immunoassay on the VITROS Immunodiagnostic and VITROS Integrated Systems.The results in Tables 1 and 2 are presented with a numerical signal-to-cutoff (S/CO) value.When comparing the S/CO values of NP samples treated with bee and wasp venoms to untreated NP samples, a noticeable decrease was observed in the S/CO values of the treated samples.www.nature.com/scientificreports/After incubation at 37 °C for 3, 6, 24, and 48 h, the NP samples treated with bee venom at a concentration of 1 mg/mL showed a significant decrease (P < 0.001) in SARS-CoV-2 antigen titer compared to the S/CO value before treatment.Similarly, the NP samples treated with bee venom at a 2 mg/mL concentration recorded lower values than the S/CO value before treatment.A high depletion rate of virus antigen titer for the concentration of 2.5 mg/mL of bee venom was recorded.Table 1 and Fig. 1 provide the numerical S/CO values and the percentage of inhibition for each time interval and concentration, demonstrating that the S/CO values of NP samples treated with bee venom were significantly different across all time intervals (P < 0.001).
For the NP samples treated with wasp venom at a concentration of 1 mg/mL, a significant decrease (P < 0.001) in SARS-CoV-2 antigen titer was observed after 3, 6, 24, and 48 h of incubation at 37 °C.The depletion rate of the virus antigen titer was higher for the concentration of 1.5 mg/mL of wasp venom, with the highest depletion rate observed at a concentration of 2 mg/mL of wasp venom compared to the S/CO value before treatment.The S/CO values of NP samples treated with wasp venom significantly differed across all time intervals (P < 0.001), except for 24 and 48 h, where the detection limit was less than 1.Table 2 displays the S/CO values for SARS-CoV-2 samples treated with wasp venom, along with the percentage of inhibition.
When comparing the concentration of 1 mg in both bee and wasp venom, it was observed that the percent of inhibition reached 97.23 ± 1.54 for wasp venom (Fig. 2), while it reached 84.1 ± 11.25 for bee venom (Fig. 1).Almost complete inhibition, around 99.72 ± 0.11, was achieved at 1.5 mg of wasp venom after 48 h (Fig. 2).In contrast, bee venom recorded a comparable inhibition percentage of approximately 98.65 ± 1.56, but at a higher concentration of 2.5 mg (Fig. 1).
The EC 90 for bee venom was determined to be 2.23 mg/mL, indicating the concentration at which 90% of its maximal antigen depletion is observed after 24-h exposure time (Fig. 3a).The EC 90 for wasp venom was 0.184 mg/ mL after 24 h (Fig. 3b).It is evident from the data that the effectiveness of wasp venom is higher than that of bee venom, and both venoms demonstrate potent efficiency in reducing antigen titer.Consequently, a cytotoxicity test was conducted on Vero E6 cells to assess the higher efficiency of wasp venom in addition to plaque reduction assay and in silico docking-based screening.

Evaluation of cytotoxicity of Vespa orientalis venom
The cytotoxicity of Vespa orientalis venom was evaluated on Vero E6 cells 48 h post-treatment using MTT assay.This finding demonstrates that the Vespa orientalis venom can affect the viability percentage of cells in a concentration-dependent manner, as shown in Fig. 4. The concentrations that inhibit 50% of the cell growth were 0.16617 mg/mL for Vero E6 cells.The MTT assay revealed the percentage of cytotoxicity, measured as the absorbance of cells with treatment compared to untreated cells, indicating a cytotoxic concentration (CC 50 ) of 0.16617 mg/mL.

3-Plaque reduction assay (SARS-CoV-2)
Table 3 presents the reduction in viral count compared to the initial viral count and the inhibition percentages for the tested wasp venom.The IC 50 of the wasp venom was determined to be 0.208 mg/mL, with the viral count at 50% inhibition recorded as 2.5 × 10 4 PFU/mL.

4-Molecular modeling of interactions of wasp venom (Mastoparan) with spike -ACE 2 of SARS-CoV-2
Molecular modeling was conducted to explore the interactions of wasp venom, specifically mastoparan, with the spike-ACE2 complex of SARS-CoV-2.Computational software was employed for molecular docking studies to assess the binding affinities of mastoparan towards the proposed target.The modeling process involved the preparation of the spike angiotensin-converting enzyme 2 (spike-ACE2), the receptor and the ligand (Mastoparan), and molecular docking principles were applied.The best-docked pose, determined by the lowest glide score value, was recorded to measure the potential interaction strength.
The molecular docking results revealed interactions of wasp venom (Mastoparan) with spike-ACE2 of SARS-CoV-2, where three poses exhibited better score values with the ability to form interactions with GLU 398, LYS 187, ASN 394, and ARG 514 in the binding pocket (Table 4).The two-dimensional interaction diagram between mastoparan and ACE2 residues illustrated the presence of four crucial hydrogen bonds (Fig. 5).The overall binding affinity was scored at -7.5.

Discussion
The limitations of current COVID-19 therapeutics necessitate the exploration of alternative and innovative therapeutic strategies. 26,27It is essential to identify novel host genes or proteins that contribute to COVID-19 pathogenesis.
Exploring animal venoms can provide valuable insights for developing new therapeutic treatments. 12.Insect venoms represent a blend of bioactive components with diverse physiological actions, rendering them promising candidates for target-specific drug discovery 12,28.Despite the completion of numerous research works on insect venom and its therapeutic potential, only a few studies have been published on the practical application of insect venom as a bioavailable drug.
Concerning COVID-19, a single published study has conducted in silico analysis of bee venom 25 ; another preprint data , which remains unpublished, has demonstrated in silico analysis of wasp venom 29 .Both studies recommend further in vitro experiments 25,29 .
However, no in vitro study has evaluated the effects of both bee and wasp venom against SARS-CoV.Additionally, both in silico analyses and cytotoxicity assays are lacking.This study aimed to fill this gap by evaluating the efficacy of both bee and wasp venom against SARS-CoV-2 in vitro and identifying the most effective one followed by in silico analysis and cytotoxicity assessment for the most potent venom.
The Ortho VITROS® Immunodiagnostic System, originally designed for clinical diagnostic purposes, has demonstrated a high correlation in measuring the antiviral activity of Hymenoptera venom in vitro.This is achieved by detecting and quantifying SARS-CoV-2 nucleocapsid antigen titers in venom-treated samples with varying concentrations and exposure times, relying on the applied S/CO values.All samples testing positive with real-time RT-PCR (Ct values below 30.0) were consistently positive in nucleocapsid antigen detection.The observed continuous decline in S/CO values with increasing venom concentration and exposure time is evidence of the anti-SARS-CoV-2 activity of Hymenoptera venom.Results indicated that wasp venom exhibited a higher antiviral effect compared to bee venom; at 48 h, the S/CO value decreased from 128.33 ± 8.74 of control nontreated samples to 0.32 ± 0.03 for 2 mg of wasp venom-treated samples, with a percent inhibition of 99.75 ± 0.03 (Table 2).In contrast, the same concentration of 2 mg bee venom, despite a lower viral load (56.63 ± 26.6) than wasp venom, resulted in a S/CO value of 5.44 ± 5.73 with a percent inhibition of 92.54 ± 7.09 (Table 1).Additionally, the 90% effective concentrations (EC 90 ) of wasp venom causing a 90% antigen depletion effect was recorded as 0.184 mg/mL, which is less than 12 times the concentration required by bee venom (2.23 mg/mL) to achieve a similar level of 90% depletion of SARS-CoV-2 nucleocapsid antigen.
The estimated IC 50 values of 223 μg/mL and 184 μg/mL for bee and wasp venom, respectively, are quite high compared to the reported 10 ng/ml IC 50 for Oriental hornet venom against Hepatitis C virus 24 .However, the antiviral potency can vary significantly depending on the specific virus and target cell types used in the assays.The high IC 50 values observed for bee and wasp venoms may be specific to the viruses and cells tested in this study especially, the lower concentrations below the tested ones didn't give any inhibition even after prolonged incubation of 72 h and no previous guide studies of antiviral activity of any crude venom or venom bioactive molecules against SARS-CoV-2 is available except Al-Rabia, et al., 2021 30 which use of Sitagliptin (SIT) with melittin (MEL) nano-conjugate against SARS-CoV-2 virus (note that, Sitagliptin (SIT) belongs to the drug class dipeptidyl peptidase-4 (DPP-4) inhibitor, medication for Type 2 diabetes.Also, Melittin is the major peptide representing approximately 50% of the dry weight of bee venom) 30 , SIT-MEL nano-conjugates showed a potent antiviral effect against the SARS-CoV-2 virus with IC 50 = 8.43 µM, the value of 8.43 µM equal to 0.01686 mg/ml meaning that SIT-MEL nano-conjugates more potent 10 times than the crude wasp venom (more potent crude venom), this difference is normal and isn't high difference as Mellittin is the primary antiviral compound, and by isolating and concentrating it, the inhibitory potency is increased compared to the whole crude venom mixture especially when conjugated with another antiviral compound like Sitagliptin moreover, the nanoformulation of both melittin and Sitagliptin can enhance its delivery, stability, and bioavailability compared to the free forms so, the importance of separating and characterizing bioactive compounds from crude venom in the future is highlighted, as well as the potential of using nanotechnology to improve the antiviral efficacy, especially against SARS-CoV-2.
It is likely that Hymenoptera venom contains compounds that induce destruction to the SARS-CoV-2 nucleocapsid antigen, resulting in low S/CO values.Notably, wasp venom demonstrated 12 times more potent virucidal activity against SARS-CoV-2 than bee venom.This variation in antiviral activity could be attributed to the differences in venom composition between the two insect species.Mastoparan, a unique and exclusive peptide found in wasp venom, has been shown to cause pores and disrupt viral lipid envelopes 31 .Consequently, it is plausible that the observed virucidal activity against SARS-CoV-2 in wasp venom may be attributed to mastoparan.Preceding studies indicate that mastoparan analogues, specifically mastoparan-7, display broad-spectrum antiviral activity against enveloped viruses representing six distinct families as observed in in vitro assays 31,32 .
The phospholipases, another component found in Vespoid wasp crude venom, can disrupt the packing of phospholipids in various biological membranes.This disruption can lead to the formation of pores and the lysis of cells by catalyzing the hydrolysis of ester bonds in specific positions of certain phospholipids 33,34.Vespoid phospholipase A2 (PLA2) demonstrates potent cytolytic actions, indicating its potential role in the antiviral activity against HCV 23 .Mastoparans, found in the wasp venom, can stimulate PLA2 from different sources 35 .PLA2 and mastoparan may work together to induce virucidal activity by forming a complex in the wasp venom.In silico investigations have shown that mastoparan interacts with various sites on ACE2, which play roles in direct or indirect contact with the ACE2 receptor.This interaction suggests a potential blocking effect on the functional ACE2 receptor complex.
The investigated compounds, particularly wasp venom, demonstrate potential efficacy as a potent inhibitor of ACE, suggesting its possible use as a novel treatment for inhibiting COVID-19 cell entry and preventing associated inflammatory complications.MTT results indicated low toxicity of wasp venom to normal cells, making it a potential candidate for antiviral applications pending further in vivo assays.Notably, the effective dose that inhibited 50% of the virus was determined to be 0.16617 mg/mL.
The antiviral effect of crude wasp venom may be attributed to one or more common or non-common molecules or toxin complexes present in the venom, particularly mastoparan.Further studies are required to identify and characterize the active compound(s) responsible for the antiviral activity of wasp venom.

In conclusion
Further research is warranted to use standard venomics techniques to isolate, identify, and fully characterize any promising SARS-CoV-2 inhibitors discovered in venom samples.Additional in vitro experiments using lung cell lines also need to be conducted to explore how these venoms might impact viral processes.All of this will help validate the potential of wasp and bee venom to treat SARS-CoV-2 in vivo, providing a basis for developing new antiviral drugs.Given wasp venom's multiple targets and promising inhibitory effects against SARS-CoV-2 based on in silico studies, it has potential to contribute to antiviral therapies.Considering the dynamic nature of COVID-19 treatments, future research should aim to compare the findings of this study with the prevailing treatment methods at that time.

Venoms
The bee Apis mellifera and the wasp Vespa orientalis venoms were extracted at the apiculture , Plant Protection Research Institute, Dokki, Giza, Egypt, by electrical chock method according to (Mohanny,et al. 2013)  36 .Both The plot of % cytotoxicity versus sample concentration was used to calculate the concentration which exhibited 50% cytotoxicity (CC 50 ).
Plaque reduction assay (SARS-CoV2).This study employed a plaque reduction assay in Vero E6 cells to evaluate the anti-SARS-CoV-2 potential of various test compounds.The methodology adhered to the protocol established by Hayden et al. (2020)  41 .
Vero E6 cells were cultivated in six-well plates for 24 h at 37 °C to achieve optimal confluence.SARS-CoV2 stocks were diluted to yield approximately 10 3 plaque-forming units (PFU)/well, ensuring consistent viral challenge.Diluted virus was pre-incubated with varying concentrations of wasp venom (within their safe use range) for a 1 h at 37 °C, facilitating potential virus-compound interaction.The pre-incubated virus-compound mixture was inoculated onto the Vero E6 cell monolayer, followed by a 1-h incubation at 37 °C to permit viral attachment.An overlay containing DMEM supplemented with 2% agarose and the test compounds was added to each well, solidifying the mixture and preventing further viral spread.Plates were incubated at 37 °C for 3-4 days, allowing efficient viral replication and the formation of distinct cytopathic plaques indicative of productive infection.Following incubation, 10% formalin was applied for 2 h to fix the infected cells, preserving their morphology for analysis.Subsequently, 0.1% crystal violet in distilled water was used to stain the fixed cells, highlighting the formed plaques for visualization and enumeration.Untreated virus-inoculated wells served as a control , establishing the baseline level of viral plaque formation in the absence of wasp venom.Visible plaques in each well were meticulously counted, and the percentage reduction in plaque formation compared to control wells was calculated using the standard formula: % Inhibition = {(viral count "untreated"-viral count "treated")/(viral count "untreated")} × 100 Analysis: By quantifying the reduction in plaque formation compared to untreated controls, this experiment provided a quantitative assessment of the antiviral efficacy of the wasp venom against SARS-CoV2 within the Vero E6 cell model.

In silico study
The in silico study was performed to predict the synergistic effect of the most potent venom (wasp venom) against spike-ACE 2 of SARS-CoV-2.

Figure 1 .Figure 2 .
Figure 1.The inhibition in neucleocapsid protein (viral load) of SARS-CoV-2after treating by bee venom of different concentration at different interval time.

Figure 3 .
Figure 3.The EC 90 of a graded S/Co values response curve showed the concentration of the venom where 90% of its maximal antigen depletion is observed after 24 h exposure duration recording 2.23 mg/ml for bee venom (a) and 0.187 mg/ml for wasp venom (b).

Figure 4 .
Figure 4. Cytotoxicity percentage of different concentration of the wasp venom against Vero E6 cells showing Half-maximum Cytotoxic concentration (CC 50 ).

Table 1 .
Signal to cutoff (S/Co) values to sars-covid-2 samples post treated by bee venom of different concentration at interval time.Data were represented as mean ± SD.ANOVA test showed a significant difference among means (P < 0.001).

Table 2 .
Signal to cutoff (S/Co) values to sars-covid-2 samples post treated by wasp venom of different concentration at interval time.Data were represented as mean ± SD.ANOVA test showed a significant difference among means (P < 0.001).